Leukemia Research Vol, 7, No. 2, pp. 133-138, 1983. 0145-2126/83$3.00 + .00 Printed in Great Britain. c¢) 1983 Pergamon Press Ltd.

IN VITRO DIFFERENTIATION OF CHRONIC LYMPHOCYTIC LEUKAEMIA CELLS WITH A SMALL

PRE-B-LIKE PHENOTYPE

JOHN GORDON, PIERRE AMAN, HAKAN MELLSTEDT, ~" PETER BIBERFELD ~ and GEORGE KLEIN

Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden and Departments of ~'Radiobiology and :~Immunopathology, Karolinska Hospital, Stockholm,

Sweden

(Received 25 October 1982. Revised 3 December 1982. Accepted 14 December 1982)

Abstract--Neoplastic populations from three cases of chronic lymphocytic leukaemia (CLL) which had features consistent with a maturation arrest at the 'small pre-B' stage are described. The cells were small and rounded with a scanty cytoplasm and stained for/a heavy chains but not light chains intracellularly while surface immunoglobulin (Smlg) was either undeteetable or expressed sparsely on a minority of ceils. Other features included the weak expression of BI, a lack of B2, an absence of the common acute lymphoblastic leukaemia antigen (cALLA), the presence of la and a variable expression of the receptors for Fcy and C3. Successful induction of in vitro differentiation in all three of the cases allowed the identification of a sequence of events whereby ceils initially containing isolated ~ heavy chains in their cytoplasm, on commencing light chain synthesis, begin to express stable SmlgM while surplus light chain is secreted without any association with the heavy chain. Although this is followed ultimately by the secretion of intact Ig effector molecules, the export of surplus light chains is apparently maintained throughout the developmental sequence. These findings are discussed with particular emphasis on their relation to normal B-cell maturation.

Key words: Chronic lymphocytic leukaemia, B-cell differentiation, immunoglobulin synthesis, phorbol ester.

INTRODUCTION

THE EARLIEST events in B-lymphocyte differentiation are characterized by a series of im- munoglobulin (Ig) gene rearrangements commencing with the/a heavy chain genes and proceeding through the light chain genes depending on the success of the previous re- arrangements [15, 16, 19]. Pre-B cells are operationally defined as lymphocytes which phenotypically express the/a chain in their cytoplasm but not the light chain and are found in foetal tissue [4], adult bone marrow [17] and represented by a variety of neoplastic analogues of both human and murine origin [8, 24, 31, 38]. As many as 20°70 of the non-T acute lymphoblastic leukaemias (ALL) have been shown to express this pre-B phenotype while even more, as many as 50070, may possess the pre-B genotype of rearranged ~ chain genes [3, 8, 38, 15].

Chronic lymphocytic leukaemia (CLL) is commonly considered to represent a neo- plastic proliferation of rather immature B lymphocytes, although clearly more differen- tiated than pre-B cells from their expression of surface Ig (Smlg) which is usually IgM often co-expressed with IgD [11, 14, 27]. In this report, however, we describe the pro- perties of the neoplastic cells from three cases of CLL taken from a large series and

*JG is a recipient of an EMBO fellowship. This investigation was also supported by PHs grant No. 1 R01 CA30264-01 awarded by the National Cancer Institute, DHHS, the Swedish Cancer Society and the Cancer Society of Stockholm.

Abbreviations: Smlg, surface immunoglobulin; CLL, chronic lymphocytic leukaemia; ALL, acute lympho- blastic leukaemia; ELISA, enzyme-linked immunosorbent assay.

Correspondence to: Dr. J. Gordon, Department of Tumor Biology, Karolinska Institute, Box 60400, 104-01 Stockholm, Sweden.

133

134 JOHN GORDON et aL

present evidence that their phenotype is consistent with a maturation arrest corresponding to the immediate progeny of the large pre-B cell and the direct precursor of true surface Ig expressing B lymphocytes.

PATIENTS AND METHODS

Patients. Three patients aged between 65 and 77 years presenting with CLL in the Stockholm area are described. The diagnosis of CLL was made on the basis of a lymphocytosis of between 40 and 154 x 109/1 of small 'well-differentiated' cells. Patient 2 was being treated with prednisolone prior to and at the time of study while the other two remained untreated apart from some intermittent leukaphoresis for patient 3. A section of enlarged turnout-infiltrated spleen was received from patient 2 within 3 h of splenectomy. Peripheral blood samples from patients 1 and 3 were processed within 1 h of collection.

Cells. Heparinized blood and finely minced tissue which had been passed through a fine wire mesh into RPMI- 1640 were centrifuged on a Ficoil-lsopaque density gradient as previously described [6]. The interface was collected and washed three times in RPMI-1640 before testing.

Immunological markers. Surface and cytoplasmic Igs were sought in direct immunofluorescent tests. Briefly, 25/~i of rabbit antibodies conjugated with FITC were added to 2 x 10 ~ pelleted cells or methanol-fixed cell smears respectively for 30 min at 0°C or room temperature respectively. The preparations were then washed and scored as described previously [6]. DAKOPATTS antibodies to human/~, 6, y, t) heavy chains and to x and ). light chains were used at a dilution of 1:10. BI, B2, cALLA, la and OKT3 positive cells were sought by indirect immunofiuorescenc¢ utilizing FITC-conjugated rabbit anti-mouse Ig (DAKOPATTS, diluted 1:10) to develop the staining. The mouse monoclonal antibodies to BI [23], B2 [22] and the J-3 antibody to cALLA [28] were the kind gift of Dr. Stuart Schlossman while mouse antibodies to la and OKT3 were obtained from New England Nuclear and Orthodiagnostics respectively. Cells beating receptors for sheep erythrocytes, Fcy and C3 were detected in rosette assays as previously described [10, 25].

Immunoglobulin secretion and stimulation studies. Freshly prepared cells were placed in RPMI-1640 at 5 x 106/ml with 10% foetal calf serum and antibiotics in a moist CO2-rich atmosphere at 37°C. When added, the phorbol ester TPA (12-0-tetradecanoyl phorbol-13-acetate) was present at 10 ng/ml while pokeweed mitogen was added at 15 pl/ml together with allogeneic T-cell factors at 50% provided from the culture supernatants of normal peripheral blood T cells incubated for three days with the mitogen. After culture with the activators for the indicated times (0-5 days), the cells were washed free of the medium and recultured for a further 18 h in fresh medium prior to the assay for extraceilular Ig.

Culture supernatants were assayed for Igs using an ELISA technique previously described [36]. In addition to the assay of~, r and ~ heavy chains and x and ~. light chains an assay specific for free light chains was developed by coating ELISA plates with DAKOPATTS antibodies specific for free r or ~. light chains (diluted l:100 and 1:150 respectively). Purified normal and/or myeloma IgM, [gG, [gA and IgD proteins did not react in the free light chain assays at levels as high as 50 pg/ml. All assays were sensitive for the appropriate proteins at levels less than i0 ng/ml.

RESULTS

In agreement with other studies [I I, 14, 27] the most common phenotype observed by us in a series of 25 cases of CLL (in preparation) was the expression of low to intermediate levels of SmlgM which was often co-expressed with IgD. This was accompanied by corres- ponding levels of the BI antigen [23] (essentially 'pan-B') and variable expression of B2 [22] (detected on more restricted B-cell types). In the three cases described here, however, SmIg was either absent (patient I) or present at threshold levels of detection on a minority of cells only (patients 2 and 3). This 'negativity' was confirmed independently during routine immunodiagnosis in a separate laboratory. All three cases were characterized by a variable number of cells which contained in their cytoplasms/~ heavy chain but no light chains as detected with fluorescein-labeIled antibodies (Table I). Within our series just less than half of the CLL patients had a neoplastic population where a variable number of cells could be found which stained in their cytoplasms for both heavy chain and mono- typic light chain equally. These cytoplasmic lg positive cases carried readily detectable quantities of Smlg. Other features of the three unusual cases described here included the sparse expression of Bl and a complete absence of B2. In none of these cases did any of the cells stain with the J-5 monoclonal antibody to cALLA. A minority population of cells only were characterized as T lymphocytes. The Ia antigen was detected on a large number of cells in both cases where it was sought while the expression of receptors for Fc), and C3 was variable and similar to other cases within our series. Fractionation of the cells on pre- generated Percoll (Pharmacia, Sweden) density gradients [26] revealed that all three

Small pre-B-like CLL

TABLE 1. IMMUNOLOGICAL MARKERS OF SMALL PRE-B CLL

135

Patient C~ Smlg BI B2 cALLA la E OKT3 Fcy C3 No. (~) (~) (~) (~) (~) (%) (~) (~) (~) (~)

l 42 0 0 0 0 45 24 12 69 17 2 90 20± 19+ 0 0 n.d. 4 n.d. 2 75 3 35 44± 37 + 0 0 59 16 14 49 23

Cells were stained for surface antigens following pre-incubation and washing at 37°C. The in- tensity of staining for Smlg and BI was judged as being either very weak (±), weak ( + ), moderate (+ +)or strong(+ + +).

n.d. = not determined.

popu la t ions con ta ined a major i ty of cells of high density. Morphologica l ly these cells were

small and rounded with a scanty cytoplasm. O n t ransfer r ing the cells to shor t - te rm culture it was demons t r a t ed that none of the

popu la t ions secreted lgM or/a chains as de termined by an E L I S A assay capable of detect- ing less than 10 ng of prote in per ml of culture supe rna tan t .

In contrast , secreted mono typ ic light chains were readily detectable in two of the cases by both the b road ly reacting and the free light chain specific ant ibodies (Table 2). The

TABLE 2. In vitro DIFFERENTIATION OF SMALL PRE-B CLL CELLS

Population Secretion Smlg Clg lgM ~ ). Free x Free ~, (%) (%)

1. Control 0 0 0 0 0 0 424a TPA 3d 43 174 0 152 0 n.d. n.d. TPA 5d 109 110 0 64 0 53 Mk + + 50 Mk

2. Control 0 0 83 0 92 20 M~t_+ 90/~ TPA 3d 495 0 285 0 128 74 M~. + 84 M~.

3. Control 0 0 80 0 80 44 M~.± 35 t~ TPA 5d 497 0 338 0 265 12 M~. + 90 M~. PWM5d 213 0 315 0 283 40 M~.+ 72M~.

Populations, either fresh or following culture with activators, were assayed for their production of extracellul_~r Ig over an 18 h period and results are presented as ng/ml/5 × 106 cells. The quantities detected in control cultures did not vary substantially over the period of testing and only the results for the first period of culture is included for simplicity. Smlg and cytoplasmic Igs (Clg) were tested following culture as described previously.

n.d. = not determined.

completely Smlg negat ive popu la t i on (pat ient 1) failed to secrete any detectable light

chain or other Ig related product over the 18 h per iod of culture. This was in fact the on ly case wi thin our series of 25 B-CLL where no light chain secret ion was evident (in p repara - t ion).

On exposure to the phorbo l ester T P A , which has been shown by others to induce the d i f fe ren t ia t ion of CLL cells in vitro [36, 37], all three popu la t ions began to produce extra- cellular mono typ ic IgM in readily detectable quant i t ies . The level of free light chain excre- t ion was either m a i n t a i n e d or, in the case of the total ly SmIg negative popula t ion , free light chain p roduc t ion was induced (Table 2). In the lat ter case bo th the induced surplus

136 JOHN GORDON et al.

light chain and the secreted IgM were associated exclusively with r light chains. This was accompanied by the appearance of readily detectable IgMk on the surface of these cells following culture with TPA but not in control cultures. Concomitant with the induction of extracellular IgM production was the appearance of cytoplasmic IgM which, in all three cases, now stained with antibodies to both g and light chains. For patient 3 a virtu- ally identical sequence of events was demonstrated after exposing the cells for 5 days to PWM together with mitogen-generated allogeneic T-helper factors (Table 2).

DISCUSSION

The majority of CLL populations apl~ar to represent a maturation arrest correspond- ing to an immature stage of B-cell development [1 I, 14, 27]. These cells have been shown to produce a surplus of light chains which are actively secreted while neoplastic analogues of more mature stages seem to produce more balanced amounts of heavy and light chains [7, 12, 20]. The extrapolation of these observations to free light chain secretion being a feature of very early B cells is in apparent contradiction to the finding that pre-B cells con- tain and synthesize only ta chains [4, 18, 31]. This anomaly could be interpreted as reflect- ing a dichotomy of developmental pathways or that CLL cells are in some way abberant in their handling of Ig molecules [20]. The present study, however, strongly implies that free light chain secreting cells can arise from precursors containing only g chains. In one case surplus light chains could be readily induced in a ~ chain only containing clone while in the other two populations free light chain secretion was demonstrated from populations where only g chains could be found intracellularly. The failure to detect intracellular light chains in those cases may reflect the rapid turnover of a relatively small light chain pool. It is pertinent to note that in the two cases where free light chain was evident prior to activa- tion these clones were expressing very low but just detectable levels of IgM at their surf- aces while in the other surface Ig could not be detected until light chain synthesis had been induced.

There is evidence from studies on a mouse lymphoma line that the expression of stable surface Ig may be associated with the onset of light chain production [24]. It is known for HLA antigens that fl2-microg!obulin is required for the transport of the HLA heavy chains through the cell [1] and it has been suggested that Ig light chains facilitate the release of the relatively insoluble heavy chain from the polyribosomes [2]. Small amounts of free light chain secretion may therefore be a normal physiological event throughout B-cell development reflecting the maintenance of a continual pool of surplus light chains in readiness for the intracellular processing of newly-synthesized heavy chains either for export or surface expression. In immature cells this secreted free light chain would mani- fest as a vast excess in the absence of significant secretion of complete Ig molecules while in high rate Ig-secreting populations, such as obtained with the TPA-driven clones, this surplus would appear as a minor contribution only. Free light chain secretion is clearly a normal event as shown from studies with foetal, neonatal and adult tissues [7, 13, 30, 32] while light chains can be readily detected in both the serum and urine of healthy indivi- duals [33, 35]. The present study now places free light chain secretion as a very early event in B-cell development but one which clearly proceeds that of isolated/~ chain expression. Whether the two cases of CLL already secreting light chains can truly be described as pre-B depends on the operational definition employed. Without the secretion studies the presence of cytoplasmic/~ chain in the absence of detectable light chain would have placed all three cases at the pre-B level. If, however, our definition of pre-B is the absence of light chain synthesis in p chain containing cells then only the neoplastic population from patient l can be considered bona fide pre-B.

This study once again emphasizes the potent action of the phorbol esters on early cells of the B-lineage. TOtterman e t al. established that TPA readily induced changes in CLL

Small pre-B-like CLL 137

cells consistent with differentiation such as preferential loss of IgD f rom the cell surfaces and the accumulat ion of cytoplasmic IgM [36, 37]. We have now demonstrated that T P A - activated analogues of early B cells are induced to secrete relatively large amounts o f lgM implying that the changes conferred on the cells relate to a quite late stage of matura t ion arrest. A more modest progression along the B-cell differentiation pathway has also recently been described for some ALL populations confirming that T P A can exert its effects on very early members of the lymphoid line [21]. It is becoming more and more clear that CLL and other neoplastic B lymphocytes display quite considerable differentia- tion potential and recent studies point to the value of this approach in attempting to eluci- date normal B-cell phenomena [9, 5, 29].

The CLL populat ions we have described here contrast with the common A L L many of which appear to reflect a matura t ion arrest equivalent to the large dividing pre-B cell [3, 8, 38]. Thus the CLL populations are small and rounded and do not express cALLA but do express Ia and some B1 both of which are found also on the pre-B ALL [21]. It is interest- ing to ask whether these cells carry other markers which are associated preferentially with either pre-B A L L or with CLL such as TdT [8] and mouse erythrocyte receptors [34] respectively and this is currently being investigated.

There is recent evidence that in normal ontogeny the large pre-B cell undergoes one division with the progeny matur ing to a small pre-B cell which is the immediate precursor of true Ig-expressing B lymphocytes [17]. It is this small pre-B cell which we feel is repre- sented here. Whether 'small pre-B CLL ' shows any distinct clinico-pathological features remains to be determined but the recognition that some SmIg-negative, non-T cases of CLL may represent a stage of matura t ion arrest for which neoplastic analogues in man were previously lacking should allow molecular events at this early stage of B-cell develop- ment to be more readily elucidated.

Acknowledgement--We wish to thank Dr. Stuart Schlossman for the generous gift of antibodies to B1, B2 and cALLA.

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